Patients with chronic inflammatory disorders, such as rheumatoid arthritis (RA), often suffer from severe osteoporosis and increased risk of fracture, partially due to reduced osteoblastic bone formation. We reported that mesenchymal stem cells (MSCs) of TNF transgenic (TNF-Tg) mice, a model of RA, have significantly decreased osteoblast (obl) differentiation. The molecules that mediate inhibited obl function in RA are unknown and there is no effective therapy. To identify new molecules in MSCs that are responsible for obl inhibition, we performed a pathway analysis using data from RNA sequencing in purified MSCs of TNF-Tg mice and WT littermates. We found that the pathway involved in Notch is markedly up-regulated in TNF-Tg MSCs. In our preliminary study, we confirmed increased Notch in MSCs from TNF-Tg RA mice and RA patients. We found that intermittent Notch inhibition prevented bone loss in TNF-Tg mice and TNF activates Notch via non- canonical NF-?B p52&RelB protein. Based on these new findings, we hypothesize that in RA MSCs, TNF activates Notch signaling through non-canonical NF-?B p52&RelB, leading to a blockade of obl differentiation. Intermittent Notch inhibition prevents bone loss in TNF-Tg mice while continuous Notch inhibition exacerbates bone loss due to depletion of the MSC pools. These hypotheses will be tested in 3 specific aims. In aim 1, we will determine if CD45-Hes1+ cells from Hes1-GFP mice functions as Notch-reporter MSCs. We will examine changes of function and Notch activation status in Hes1+MSCs from different ages of Hes1-GFP/TNF-Tg mice during the course of RA development. In aim 2, we will use biochemical and molecular approaches to study mechanisms by which canonical NF-?B p52&RelB, as well as canonical NF-?B p50&RelA mediate the effect of TNF on Notch activation and if this is through the canonical Notch signaling pathway. In aim 3, we will determine if intermittent, but not continues, administration of Notch inhibitor rescues bone loss phenotype of TNF-Tg mice and if increased Notch activation and inhibited obls in TNF-Tg mouse MSCs are mediated by TNF using TNF inhibitor. Our study will reveal a novel role for Notch in mediating RA-associated bone loss and interaction between non-canonical NF-?B and Notch, and generate preclinical information on Notch inhibitors as a new potential therapy for treating patients with RA or other chronic inflammation-associated osteoporosis.